We will determine the relationship of two beta-actin mutations to abnormal cytoarchitecture, elevated growth potential, and neoplastic transformation of human fibroblasts. This is to be accomplished in the following steps: (1)\cloned genes for two mutant and the one functional wildtype beta-actin will be isolated from gene libraries of HUT-14 and HUT-14T cells; these actin genes will be specifically selected by recombination of the actin gene clone in lambda DNA with a specially constructed plasmic PiVX containing a sub-cloned portion of beta-actin cDNA homologous to the 3' untranslated region of the gene; (2)\mutant and wildtype genes will be distinguished by their sensitivity to a restriction enzyme at the mutation site coding for amino acid 244, where a Gly is exchanged for an Asp; (3)\mutant actin genes will be sequenced in their coding regions to confirm the mutation at codon 244 and to identify the site of the second mutation in the mutant beta-actin gene derived from HUT-14T; (4)\these actin genes will be co-transferred with pSV-2 plasmid, which has the SV40 early transcription promoter and the E. coli gpt gene, into normal human and rodent fibroblasts using the calcium phosphate precipitation technique; (5)\when actin genes are coupled to the gpt genes, HPRT-deficient normal fibroblasts and preneoplastic fibroblasts will be the recipients for gene co-transfer using selective (HAT) medium and cultivated to allow progressive expression or elevation of tumorigenic potential; and (6)\cultures treated with actin genes will be assessed for alterations in phenotype and transformation by monitoring mutant beta-actin synthesis, escape from senescence, focus formation, anchorage independence, aneuploidy, protein profiles in two-dimensional gels, protease expression, and tumorigenesis in athymic mice. This approach should test the involvement of these actin mutations in abnormal cytoskeletal structure and function and expose their relationship to neoplastic transformation and tumorigenicity. (L)